Separating agent comprising bonded conalbumin

ABSTRACT

Disclosed is an optical isomer separating agent composed of a stationary phase having a support and a chemically modified conalbumin. The separating agent may be used in a high-performance liquid chromatography column.

FIELD OF THE ART

The present invention relates to an optical isomer separating agentwhich comprises a support and conalbumin bonded thereto and to ahigh-performance liquid chromatographic column packed with theseparating agent.

BACKGROUND ART

It is known that only one of the optical isomers constituting a racemicmodification has a physiological activity in the natural world. It hasrecently been found in the field of drugs that only one of the opticalisomers constituting a racemic modification has a remarkablepharmacological effect or a low toxicity in some cases, for which thedevelopment of a drug with an isolated optical isomer has risen inimportance.

Only a few methods for the separation of optical isomers can bepractically conducted on an industrial scale, though many methodstherefor have been known hitheroto. Therefore, a method which canseparate optical isomers easily and at a low cost has been sought. Inrecent years, with the progress in high-performance liquidchromatography, methods for separating optical isomers have become knowngenerally and separatory columns having various performances have alsobeen reported.

However, there are only a few columns usable for the separation ofoptical isomers while it is known that very many compounds have opticalisomers. In addition, not all optical isomers can always be efficientlyseparated, and therefore the development of a new separatory column hasbeen expected.

DISCLOSURE OF THE INVENTION

The present inventors have extensively studied to develop an opticalisomer separating agent having a new separating ability. As a result,they have found that the above object can be attained by taking thefollowing constitution. The present invention has been accomplished onthe basis of this finding.

Namely, the present invention relates to an optical isomer separatingagent characterized by being composed of a stationary phase comprising asupport and conalbumin or chemically modified conalbumin bonded to thesupport. Further, the present invention also relates to ahigh-performance liquid chromatographic column packed with an opticalisomer separating agent characterized by being composed of a stationaryphase comprising a support and conalbumin or chemically modifiedconalbumin bonded to the support.

According to the present invention, optical isomers which could not beseparated or could only be insufficiently separated according to theprior art can be efficiently separated. Accordingly, an object of thepresent invention is to provide a new optical isomer separating agentwhich has a new optical isomer separating ability and a high-performanceliquid chromatographic column packed with this new separating agent.

The support in the present invention refers to a fine support to whichconalbumin can be chemically bonded, and examples thereof include silicagel, glass, cellulose, synthetic polymers and aminopropyl silica gel.

Conalbumin is a protein falling under the category of albumins and itsmolecular weight is 70,000 (or 87,000 according to some other reports).The pH of the isoelectric point thereof is 5.8. It exists in egg whiteand accounts for 13.8% of the proteins constituting the egg white.

The conalbumin used may be a commercially available one. Alternatively,the conalbumin may be prepared from egg white by a known process (KagakuDaijiten (Encyclopaedia Chimica), Vol. 3, p.744 (1977)).

Conalbumin can be bonded to a support by a conventional process for thepreparation of a stationary phase. For example, when aminopropyl silicagel is used as the support, the bonding of the conalbumin can beconducted by using N,N-disuccinimidyl carbonate as a crosslinking agent.Further, when silica gel is used as the support, the bonding of theconalbumin can be conducted by using 3-glycidoxypropyltrimethoxysilaneas a crosslinking agent.

The chemically modified conalbumin in the present invention refers toconalbumin which is partially chemically converted by cross-linkage withglutaraldehyde, conversion into diol, acylation or modification withglutaraldehyde followed by reduction. Methods for chemical modificationare as follows: for example, the cross-linkage of conalbumin withglutaraldehyde is conducted by adding conalbumin and glutaraldehyde to aphosphate buffer having a pH of 6.8 and stirring the obtained mixture at30° C. for 15 hours, by which conalbumin cross-linked withglutaraldehyde is obtained. The conalbumin cross-linked withglutaraldehyde is further treated with sodium borohydride in a phosphatebuffer having a pH of 6.8 at 4° C. for 12 hours under stirring to give areduced-form conalbumin. Separately, N,N-disuccinimidyl carbonate isdissolved in acetonitrile, followed by the addition thereto of a sodiumhydrogencarbonate buffer and aminopropyl silica gel to effect areaction. After the organic solvent was washed off, the reaction productis reacted with the above chemically modified conalbumin in a sodiumhydrogencarbonate buffer to give a support containing the chemicallymodified conalbumin bonded thereto.

Alternatively, a support containing a chemically modified conalbuminbonded thereto can also be prepared by chemically modifying a supportcontaining conalbumin bonded thereto.

The optical isomer in the present invention refers to a chiral compoundhaving an asymmetric carbon atom in its molecule, which includes manycompounds. Examples of such a chiral compound which is used in the fieldof drugs include clorprenaline, ascorbic acid, ampicillin, tocopherol,epinephrine, ephedrine, quinine, phenylephrine, propranolol,methamphetamine, scopolamine, methyldopa, azelastine and verapamil.

The optical isomer separating agent according to the present inventionis generally used by packing the agent into a column and attaching theresulting column to a high-performance liquid chromatograph.Accordingly, the separation of optical isomers may be conducted byconventional high-performance liquid chromatographic processes underanalytical conditions including the composition of the mobile phasebeing suitably selected depending upon the properties of the objectivesubstance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the liquid chromatogram of d,l-azelastine obtained by usingthe column according to the present invention; FIGS. 2 and 3 show theliquid chromatograms of d,l-azelastine obtained by using an ovomucoidcolumn; and FIG. 4 shows the liquid chromatogram of d,l-verapamilobtained by using the column according to the present invention. In theFIGS. 1 and 4, each left peak is assignable to d-isomer, while eachright peak to l-isomer thereby exhibiting the distinct separation ofeach enantiomer.

BEST MODE TO PRACTICE THE INVENTION

The present invention will be described in more detail by referring tothe following Examples, though the present invention is not limited tothem.

EXAMPLE 1

3 g of aminopropyl silica gel and 2 g of N,N-disuccinimidyl carbonatewere added to 100 ml of a 0.1M sodium hydrogencarbonate buffer (pH 6.8).The obtained mixture was stirred overnight and poured on a glass filter.The filter cake was washed with water and a suspension of activatedaminopropyl silica gel was prepared. A solution of 2 g of conalbumin in30 ml of a 0.1M sodium hydrogencarbonate buffer (pH 6.8) was separatelyprepared and added to the above suspension to give a separating agentaccording to the present invention.

EXAMPLE 2

The separating agent prepared in Example 1 was packed into a steelcolumn to give a separatory column for optical isomers.

EXAMPLE 3

10 g of silica gel was dried at 140° C. for 24 hours, cooled andsuspended in 140 ml of toluene, followed by the addition thereto of 15ml of 3-glycidoxypropyltrimethoxysiline. The obtained mixture wasrefluxed with heating. After 5 hours, low-boiling substances weredistilled away from the top and the resulting mixture was poured on aglass filter. The filter cake was washed with toluene, tetrahydrofuranand methanol successively and dried at 60° C. for 2 hours to give anepoxy-activated silica gel. 5 g of this activated silica gel wassuspended in 50 ml of a borate buffer having a pH of 8.5, followed bythe addition thereto of 500 mg of conalbumin. The obtained mixture wasreacted at room temperature for 24 hours and poured on a glass filter.The filter cake was washed with water to give a separating agentaccording to the present invention.

EXAMPLE 4

The separating agent prepared in Example 3 was packed into a column togive a separatory column for optical isomers.

EXAMPLE 5

2 g of the support prepared in Example 1 and 0.1 g of glutaraldehydewere added to 30 ml of a 0.06M phosphate buffer (pH 6.8). The obtainedmixture was stirred at 30° C. for 15 hours to form a support containing,bonded thereto, conalbumin (unreduced form) cross-linked withglutaraldehyde. Further, 0.2 g of sodium borohydride was added to theabove reaction mixture, and the resulting mixture was stirred at 40° C.for 12 hours to give a support containing, bonded thereto, conalbumin(reduced form) reduced and cross-linked with glutaraldehyde.

EXAMPLE 6

The support prepared in Example 5 was packed into a column to give aseparatory column for optical isomers.

Effect

The effect of the present invention will be described by referring tothe following Experimental Examples.

EXPERIMENTAL EXAMPLE 1

The separation of azelastine was conducted by the use of the separatorycolumn for optical isomers prepared in Example 2.

Conditions of high-performance liquid chromatography

mobile phase: 50 mM phosphate buffer (pH 5.0) containing 8% of ethanol

flow rate: 1.0 ml/min

detection: 230 nm

The result is shown in FIG. 1.

The peak at a retention time of 19 minutes is assignable to the d-isomerand that at a retention time of 25 minutes to the l-isomer. As apparentfrom the FIG. 1, the optical isomers of azelastine were separated by theuse of the separating agent according to the present invention.

COMPARATIVE EXAMPLE 1

The separation of the optical isomers of azelastine was conducted by theuse of a commercially available ovomucoid column (trade name: ULTRONES-OVM) using ovomucoid, which is a glycoprotein. FIGS. 2 and 3 show thechromatograms obtained by using 2% and 1% tetrahydrofuran/20 mMphosphate buffer (pH 4.8) as the mobile phase, respectively.

As apparent from FIGS. 2 and 3, the optical isomers of azelastine couldnot be separated. By using methanol or acetonitrile as the mobile phase,the optical isomers of azelastine could not be separated as well.

EXPERIMENTAL EXAMPLE 2

The separation of verapamil was conducted by the use of the separatorycolumn for optical isomers prepared in Example 2.

Conditions of high-performance liquid chromatography

mobile phase: 50 mM phosphate buffer (pH 4.5) containing 5% of ethanol

flow rate: 1.0 ml/min

detection: 230 nm

The result is shown in FIG. 4.

The enantiomers are eluted at a retention time of 2.5 minutes for thed-isomer, and a retention time of 3.5 minutes for the l-isomer. As isapparent from FIG. 4, the optical isomers of verapamil can be separatedby the use of the separating agent of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the liquid chromatogram of d,l-azelastine obtained by usingthe column according to the present invention; FIGS. 2 and 3 show theliquid chromatograms of d,l-azelastine obtained by using an ovomucoidcolumn; and FIG. 4 shows the liquid chromatogram of d,l-verapamilobtained by using the column according to the present invention. In theFIGS. 1 and 4, each left peak is assignable to d-isomer, while eachright peak to l-isomer thereby exhibiting the distinct separation ofeach enantiomer.

We claim:
 1. An optical isomer separating agent characterized by being composed of a stationary phase comprising a support and a conalbumin which is partially chemically converted by cross-linkage with glutaraldehyde, conversion into a diol, acylation or modification with glutaraldehyde followed by reduction. 